Whooping cough is the childhood disease caused by the Gram negative bacterium, Bordetella pertussis. It was thought that this disease was being managed adequately by the current immunization program, but the safety of the pertussis vaccine has been recently questioned. Development of a safe replacement vaccine will require an understanding of the disease process, in particular, determining the role of the bacterial components, in the disease and as antigens. Our long term goal is to understand the molecular basis of whooping cough. Virulent B. pertussis organisms produce multiple virulence factors including several toxins. The virulence factors are all coordinately expressed under some growth conditions, and are all repressed under others. We propose to use this property to isolate mutants deficient in virulence factors using Tn5 lac, a derivative of Tn5, which fuses expression of beta galactosidase to exogenous promoters. We will screen for mutants in virulence genes by screening for kanamycin resistant Tn5 lac insertion mutants that express beta galactosidase under conditions where the virulence factors are expressed, but not when synthesis of the virulence genes is turned off. The phenotype of these mutants will be determined and the importance of the missing virulence factor will be assessed by determining the ability of the mutant to cause disease in the infant mouse model. This new methodology will increase our understanding of the disease process. This information is crucial for the development of a safe replacement vaccine.